The c-fes proto-oncogene encodes the cytoplasmic protein-tyrosine kinase FES, which participates in cellular signaling cascades that govern differentiation, survival, migration and inflammation. A recent kinome-wide siRNA screen identified FES as essential for the growth and survival of human multiple myeloma (MM) cell lines. FES is also a common signaling mediator for several angiogenic factors, making FES a potential target for anti-angiogenic therapy in MM and other cancers. Recently, we reported the discovery of first-in-class small-molecule FES kinase inhibitors with nanomolar potency. Using these inhibitors, we established a new role for FES kinase activity in osteoclast differentiation from mouse bone marrow macrophages, suggesting that inhibition of this FES-dependent pathway may be of clinical benefit in osteolytic bone loss associated with MM. Together, these observations suggest that inhibition of FES kinase activity may have a three-pronged benefit in MM: 1) direct inhibition of myeloma tumor cell growth; 2) block in MM-driven osteoclast differentiation; 3) suppression of tumor angiogenesis. Here we propose to validate FES as the target for our inhibitors in each of these aspects of MM with the following Specific Aims: Aim 1: Test the hypothesis that the FES protein-tyrosine kinase is a tumor-intrinsic drug target in multiple myeloma. Although RNAi-mediated knockdown of FES expression induces apoptosis in several human myeloma cell lines, the frequency with which FES protein expression and kinase activity are upregulated in MM has not been explored. We propose to determine FES expression and activity profiles for a diverse panel of human MM cell lines, patient- derived MM cells and myeloma tissue microarrays. MM cells will then be tested for sensitivity to our FES inhibitors in terms of cell proliferation and survival. Validation of FES as the inhibitor target wll involve rescue of inhibitor sensitivity by ectopic expression of engineered inhibitor-resistant FES mutants in sensitive cells. Conversely, introduction of active FES into inhibitor-insensitive, FES-negative MM cells may render them sensitive to these compounds. Aim 2: Test the hypothesis that FES kinase activity is required for MM-associated osteolysis and angiogenesis. Recently we made the unexpected discovery that multiple classes of FES kinase inhibitors potently block osteoclast differentiation from primary mouse bone marrow macrophages and cell lines. We propose to test our Fes inhibitors for suppression of osteoclast formation from primary human macrophages, and determine whether the inhibitors also suppress their osteolytic activity in bone resorption assays. In addition, we will use our FES inhibitors to explore the role of FES kinase activity in endothelial cell proliferation, migration and capillary formation using human umbilical vein endothelial cells (HUVECs). Finally, we will evaluate the consequences of FES inhibition on MM cell adhesion to vascular endothelial cells using a MM/HUVEC co-culture system.